PROJECT SUMMARY In its 2015 report, the World Health Organization declared that tuberculosis (TB) killed more humans than any other infectious agent. While treatment of drug susceptible TB requires combination therapy for at least six months, treatment of multidrug-resistant TB (DR-TB) requires treatment with multiple toxic, expensive and less efficacious second/third line drugs for up to 2 years. Even then, only ~50% of DR-TB cases that receive treatment have successful outcomes. In a recent study, 12% of treated DR-TB cases developed additional drug resistance during treatment. Therefore, new drugs and innovative regimens that are effective against drug-resistant TB are urgently needed. Development of new drugs and regimens is a core mission of our Center for Tuberculosis Research and our longstanding industry partner, the TB Alliance. Over the past decade, our Pre-clinical Regimen Identification Program identified 7 novel drug combinations containing 2-3 drugs unapproved for TB treatment that subsequently advanced to clinical trials. TB Alliance-funded phase 3 trials are underway (n=2) or being planned (n=1) for 3 regimens, all of which include MDR-TB patients. Yet, there remains a great need for potent new, oral agents for safer and more universally active regimens without drug-drug interactions. Clinical proof-of-concept for TB therapy was recently demonstrated for the carbapenem meropenem in combination with amoxicillin/clavulanate. We have developed a series of chemically distinct synthetic penems, with superior whole cell potency over existing carbapenems, owing to their being optimized for inhibition of non-classical L,D-transpeptidases that comprise the predominant transpeptidase activity during the final step of peptidoglycan synthesis in M. tuberculosis. We determined the crystal structure of one of our lead penems (T402) bound to a M. tuberculosis L,D-transpeptidase and proposed a unique mechanism of action. Structural differences were engineered to enhance potency and selectivity against M. tuberculosis and to minimize spontaneous resistance. This proposal describes a comprehensive, milestone-driven pre- clinical development plan with the principal objective of delivering a novel penem and novel penem- containing regimens ready for IND-enabling toxicity studies and further clinical development for treatment of DR-TB.